Nuclear entry, oligomerization, and DNA binding of the Drosophila heat shock transcription factor are regulated by a unique nuclear localization sequence.

Abstract

In normally growing Drosophila cultured cells the Drosophila heat shock transcription factor (dHSF) is localized in the cytosol and translocates into the nucleus after heat shock. In the cytosol of nonshocked cells, the dHSF is present as a monomer that cannot bind DNA. Upon stress, the dHSF enters the nucleus where it is observed to be a trimer. A novel nuclear localization sequence (NLS) in the dHSF was found to be responsible for stress-dependent nuclear entry. Deletion of the NLS prevents nuclear entry, as expected, yet surprisingly also allows constitutive oligomerization and DNA binding in the cytosol. Further analysis of the NLS by mutagenesis suggests that the two functions of nuclear entry and oligomerization are separable in that distinct residues present in the NLS are responsible for each. Mutations in certain basic residues completely block nuclear entry, as expected for a constitutive NLS. In addition, two residues were found in the NLS that, when altered, allowed constitutive nuclear entry of dHSF independent of stress. These residues may interact with a putative cellular component or possibly other domains of the HSF to prevent nuclear entry in normally growing cells. The NLS can also function autonomously to target a 13-galactosidase fusion protein into the nucleus in a heat shock-dependent fashion.[Key Words: Transcription regulation; nuclear localization, DNA-binding regulation]Received November 25, 1996; revised version accepeted March 21, 1997.The activity of specific transcription factors can be regulated at several levels, including DNA binding and transcription activation (Keegan et al. 1986; for review, see Ptashne 1988). Regulation of DNA binding is often the result of cytoplasmic sequestration through association with other proteins (for review, see Baeuerle and Henkel 1994}. For example, steroid receptors are localized in the cytoplasm in association with other proteins that restrict their entry into the nucleus (Picard and Yamamoto 1987; for review, see Hanover 1992). When the appropriate hormone ligand is present, these complexes are disrupted and the receptor can enter the nucleus to activate transcription (Picard and Yamamoto 1987). Similarly, the NF-KB/rel/dorsal family of transcription factors is tethered in the cytosol by association with IKB proteins (Baeuerle and Baltimore 1988;Steward 1989;

A Beta-Stranded Motif Drives Capsid Protein Oligomers of the Parvovirus Minute Virus of Mice into the Nucleus for Viral Assembly

“…Remarkably, some of the conserved basic residues of the NLM of MVMi seem to be involved additionally in interactions with DNA (mutant ⌬VP-1/VP-2 K530N-R534 [ Fig. 3A]), a property previously described in some NLSs (62,76). The analysis of the ordered DNA in the canine parvovirus capsid structure (68) showed a low level of specificity associated with the interaction between the DNA bases and the capsid protein, which may contribute to DNA packaging during viral assembly (12).…”

The Product of the Rice myb7 Unspliced mRNA Dimerizes with the Maize Leucine Zipper Opaque2 and Stimulates Its Activity in a Transient Expression Assay

“…Several nuclear proteins, and among them some Myb factors, do not show any canonical consensus for nuclear localization (62)(63)(64). Some of these enter the nuclei only upon interaction with their partner, like APETALA and PISTILLATA (65), and Dp and E2F (66).…”

Regulation of IκB Kinase Complex by Phosphorylation of γ-Binding Domain of IκB Kinase β by Polo-like Kinase 1

“…Electrophoretic Mobility Shift Assay (EMSA)-Whole cell lysates (4 g) were incubated with labeled NF-B or Oct-1 (octamer factor 1) oligonucleotides (25,28) in gel shift mixture containing the following: 4% Ficoll-400, 1 mM EDTA, 1 mM DTT, 50 g/ml poly(dI-dC), and 25 g/ml single-stranded DNA. The complexes were resolved on a 6% native polyacrylamide gel and analyzed by a PhosphorImager (GE Healthcare).…”